The cell cycle is normally regulated by cyclin-dependent kinases (CDKs), a family of serine/threonine kinases activated by a family of cyclins. The first identified CDK, cdc2 was discovered in the yeast; its human homolog, CDK1, was soon identified and confirmed to be present in all eukaryotes investigated. The 34-kDa protein homologous to the product of the cdc2 gene of the fission yeast is also called p34cdc2. Cyclin B was discovered in sea urchin oocytes and was demonstrated to belong to a family of several members that can be found in a wide variety of cells. CDKs complexed with cyclins play a role not only in the regulation of the cycle of cell division but also in apoptosis, and in the control of transcription (reviewed in ref. 25).
As with mitosis in other cells, meiosis in oocytes is initiated by the M-phase-promoting factor (MPF), which is a heterodimer of p34cdc2 and cyclin B. During interphase, cyclin B accumulates and binds to p34cdc2. At this point, phosphorylation of p34cdc2 inhibits the function of the complex, whereas subsequent dephosphorylation by the phosphatase cdc25 provides p34cdc2 with kinase activity, leading to the generation of active MPF and entry into first metaphase. In mammals, the meiotic arrest at metaphase II after ovulation is also sustained by high MPF activity that is maintained through an equilibrium of continuous synthesis and degradation of cyclin B.
MPF is stabilized by the cytostatic factor, which is composed of at least three proteins: Mos, mitogen-activated protein (MAP) kinase, and p90Rsk (4). In general, these proteins are responsible for maintaining the condensed status of the chromatin during the meiotic arrest.
After sperm-egg interaction, the activity of these cell cycle-related regulatory proteins decreases remarkably. The increase in the intracellular free calcium concentration at fertilization triggers the activation of calmodulin-dependent protein kinase II, which in turn triggers the degradation of cyclin B which, together with p34cdc2 phosphorylation, results in the inactivation of MPF. Low MPF activity will stimulate the resumption of meiosis, chromatin decondensation, and entry into interphase. The activation of calmodulin-dependent protein kinase II also induces cytostatic factor inactivation (26). MAP kinase activity also decreases after oocyte activation: in the mouse, the dephosphorylation of MAP kinase results in its inactivation that correlates with the formation of the nuclear envelope and initiation of DNA synthesis (27).
During parthenogenetic activation of young bovine (28) and rabbit (29) oocytes, MPF activity rapidly recovered after an activating stimulus, which allowed recondensation of chromosomes and re-entry of the oocytes into a new M-phase arrest known as metaphase III. This arrest can be circumvented by treatments that are able to inhibit MPF (and possibly MAP kinase) activity.
3.2.1. Inhibiting MPF Activity With Protein Kinase Inhibitors
Inhibiting protein kinases to dephosphorylate proteins in general seems to trigger oocyte activation. Nuclear lamins are proteoglycans that constitute the nuclear envelope and are thought to facilitate the overall organization of the nucleus. Their dephosphorylated state corresponds to the formation of the nuclear envelope and, when phosphorylated by the p34cdc2/cyclin B complex at the G2/M transition, nuclear lamins are disassembled. Furthermore, a cyclic adenosine monophosphate-dependent kinase inhibitor seems to localize in the nucleus of G2/M cells. Its inhibition results in the arrest of the cell cycle indicating that protein kinase inhibition may be a normal function for the M/G1 transition (30). Inhibition of protein kinases with the nonspecific kinase inhibitors staurosporine or H7 results in the resumption of meiosis and formation of pronuclei in mouse and porcine oocytes (31,32). Inhibition of MAP kinase activity, independently of MPF inactivation using U0126, a MAP kinase inhibitor also induced some degree of activation in porcine oocytes (33). Finally, inhibition of myosin light-chain kinase activity may partially be responsible for activation stimulated by kinase inhibition. It was demonstrated that blocking myosin light-chain kinase activity eventually releases the cyclin degradation machinery resulting in pronuclear formation in porcine oocytes (34).
As mentioned previously, dephosphorylation of the p34cdc2-cyclin B complex generates active MPF that is involved in maintaining the meiotic arrest. The dephosphorylation is catalyzed by the phosphatase cdc25, whose activity can be blocked by inhibiting its phosphorylation by protein kinase inhibitors. In Xenopus laevis oocytes the compound 6-DMAP, a broad-spectrum kinase inhibitor, causes release from the meiotic arrest by inhibiting cdc25, thus preventing dephosphorylation (i.e., activation) of MPF (4). It is also thought to be important in inhibiting protein phosphorylation necessary for the spindle apparatus because, in many species, 6-DMAP inhibits the extrusion of the second polar body as well (see Note 5). Applied after an induced calcium transient, it was used effectively for oocyte activation in a number of NT experiments.
The incubation of oocytes with ionomycin and subsequently, with 6-DMAP stimulates activation effectively (28) and, by means of this combined activation, live offspring have been produced in a number of species after NT. Recently ovulated (young) oocytes, although believed to be of better quality, have proved difficult to activate because of consistently high MPF activity. By preventing reactivation of MPF, 6-DMAP allows the use of younger oocytes for NT, at least in cattle.
In cattle: Incubation in 5 mM ionomycin for 4 min, then in 2 mM 6-DMAP for 3 h (35).
In sheep: 5 mM Ionomycin for 5 min, then 2 mM 6-DMAP and 7.5 mg/mL cytocha-lasin B for 1 h, followed by an incubation in 2 mM 6-DMAP for an additional 1 h (36).
In pig: 15 mM Ionomycin for 20 min followed by 1.9 mM 6-DMAP for 3 to 4 h (37).
In goat: 5-min Incubation in 5 mM ionomycin then 3 h in 2 mM
In Rhesus monkey: 5 mM Ionomycin for 2 min followed by 4 h in 2 mM 6-DMAP and 5 mg/mL cytochalasin B (39).
Oocytes activated with the combined ionomycin/6-DMAP treatment reportedly have alterations in their DNA content owing to an abnormal pattern of karyokinesis during their first cell cycle (40). In somatic cells arrested in S-phase, 6-DMAP also was shown to induce premature mitosis followed by reformation of the nuclear envelope around decondensed and fragmented chro-matin to form numerous micronuclei (41). When used to activate oocytes after NT, placental malformations and perinatal death have been reported in the resulting offspring (35). These problems might indicate that protein kinase inhibitors with low selectivity are not acting on one specific kinase only but they interfere with several metabolic pathways involved in other cell functions whose inhibition may have deleterious consequences in subsequent cellular events (4).
6-DMAP has been characterized as a broad-spectrum kinase inhibitor. The targeted inhibition of specific kinases that play crucial roles in cell cycle regulation seems to be a more attractive way to induce oocyte activation. Because naturally occurring CDK inhibitors are important in cell cycle control, artificial inhibition of these CDKs is believed to be an effective way to influence the cell cycle (25). Selective compounds for the inhibition of MPF activity were developed; butyrolactone I inhibits selectively CDK1 (p34cdc2) and CDK2, effectively arresting the cell cycle at G2/M and G1/S (42). By inhibiting cdc2, the catalytic subunit of MPF, butyrolactone I was described to inhibit meiotic resumption in GV stage oocytes or to induce activation in oocytes that are arrested in the MII stage (43). A combined electrical and butyrolactone I treatment resulted in high rates of pronuclear formation and cleavage in porcine oocytes (44), and butyrolactone I after an ionomycin-induced calcium transient has been reported to stimulate development to term in bovine NT experiments (45).
In cattle, expose NT bovine oocytes to 5 ^M ionomycin for 4 min, and then transfer them into 100 ^M butyrolactone I for a 4-h culture (45).
3.2.2. Inhibiting MPF Activity With Protein Synthesis Inhibitors
To maintain MPF activity, cyclin B must be synthesized continuously. Blocking the production of cyclin B with protein synthesis inhibitors was shown to stimulate oocyte activation. Inhibitors such as puromycin or cycloheximide could induce entry into the first interphase in mouse and human but not in rat and pig oocytes (reviewed in refs. 3 and 4).
Inhibition of protein synthesis is possible by exposing the oocytes to cycloheximide, an antifungal antibiotic, for an extended period of time. The treatment was reported to activate rhesus monkey and bovine oocytes after NT.
In Rhesus monkey: Treat NT oocytes with 7.5 ||g/mL cycloheximide and 7.5 ||g/mL cytochalasin B for 1 h. Fuse subsequently with an electrical pulse in the presence of 100 | M calcium which may provide an additional activating stimulus (46).
In cattle: incubate reconstructed oocytes in 10 | g/mL cycloheximide and
5 |g/mL cytochalasin B for 1 h, then in cycloheximide alone for an additional 4 h (47).
Activation and subsequent development has generally been more successful when the inhibition of protein synthesis is preceded by a stimulus that triggers a calcium transient (48). As mentioned previously, the efficiency of partheno-genetic oocyte activation is age dependent with freshly ovulated oocytes being more difficult to activate. Similarly to protein kinase inhibition, blocking protein synthesis in combination with an induced calcium release caused activation of bovine oocytes that was independent of age (49).
After an electric pulse, cycloheximide reportedly triggered term development of NT embryos of various species.
In cattle: Two DC pulses of 250V/mm for 10 |is each, followed by a culture in 10 |g/mL cycloheximide for an additional 5 h (50).
In goat: A single pulse of 100 V/mm for 160 |is, then culture in the presence of 7 |g/mL cycloheximide for 3 h (51).
In cat: Two 100 V/mm, 50-|is pulses, 5 s apart. Subsequently, incubation for 6-7 h in 10 |g/mL cycloheximide and 5 |g/mL cytochalasin B (52).
Rabbit: Three DC pulses of 320 V/mm for 20 | s each; a second set of pulses 1 h later. Then incubation in 5 | g/mL cycloheximide and 2 mM 6-DMAP for 1 h (53).
126.96.36.199. Ethanol Plus Cycloheximide
When used in combination with ethanol, cycloheximide was successfully used after bovine NT.
Application: In cattle, incubate reconstructed bovine oocytes in 7% (v/v) ethanol for 5 min and then culture for 5 h in 10 |g/mL cycloheximide and 5 |g/mL cytochalasin B. (54).
188.8.131.52. A23187 Plus Cycloheximide cycloheximide also was applied in combination with the calcium ionophore A23187 for the production of cloned calves.
Application: After NT in cattle, expose the reconstructed oocytes to 5 |M A23187,5 |g/mL cytochalasin B, and 10 |g/mL cycloheximide for 10 min, then incubate in 10 |g/mL cycloheximide and 5 |g/mL cytochalasin B for 1 h. Follow by culture in the presence of 10 |g/mL cycloheximide for an additional 5 h (55).
184.108.40.206. Ionomycin Plus Cycloheximide cycloheximide treatment after a calcium transient induced by ionomycin was reported to induce term development of ovine and horse oocytes after NT.
In sheep: Incubate oocytes with 5 ||M ionomycin for 5 min and, subsequently, in 10 | g/mL cycloheximide and 7.5 | g/mL cytochalasin B for 5 h (56).
In horse: Enucleated equine oocytes were fused with donor cells isolated from a mule fetus and cultured in 5 | M ionomycin for 5 min, followed by a 5-h culture in 10 | g/mL cycloheximide. During activation, the calcium concentration in the medium was markedly higher (4.26 mM) than reported for other species (57).
The inhibition of protein synthesis with cycloheximide is widely used for oocyte activation after NT. It was reported, however, that besides depleting the oocytes of proteins that maintain MPF activity cycloheximide also inhibits translation of proteins responsible for the initiation of DNA replication (58). In NT embryos activated by ethanol/cycloheximide the initiation of DNA synthesis was delayed (59), and a great percentage of offspring had various health-related problems (54).
In certain cases, the recipient oocytes are activated before the donor nucleus is introduced into the cytoplasm. This process is called preactivation and is applied primarily when blastomeres are used as donor cells for NT. The rationale behind preactivation is the necessity of cell cycle synchronization between the two cells. In cattle, it was demonstrated that most blastomeres are in the S-phase of their cell cycle, and activation of the recipient cytoplasm approx 4 h before fusion will induce MPF levels to decrease and the oocyte will be in the S-phase by the time the donor nucleus is transplanted into the cytoplasm (60). During somatic cell NT, when the donor cells are generally synchronized in the G0 or Gx stage of their cell cycle, better development can be expected if the recipient cytoplasm is at the metaphase stage. To achieve this, the oocytes should be activated at the time of fusion or some time thereafter.
1. The effectiveness of an oocyte activation method can be measured by how efficiently it can stimulate the initiation of the oocyte developmental program. Before being used for activation during a NT experiment, each method should be tested; pronuclear formation and development to the blastocyst stage provide convenient end points for an initial evaluation.
2. The electroporation medium typically contains a very low amount of electrolyte; the main component is a sugar such as sucrose or mannitol. The Zimmermann fusion medium (0.28 M sucrose, 0.5 mM Mg(CH3CO2)2, 0.1 mM Ca(CH3CO2)2, 1 mM K2HPO4, 0.1 mM gluthatione, and 0.01 mg/mL bovine serum albumin ) is probably the most established medium used for electroporation after NT. We found that a medium consisting of 0.3 M mannitol, 0.1 mM CaCl2, 0.1 mM MgSO4, 0.5 mM HEPES, and 0.01 mg/mL bovine serum albumin also works very sufficiently, at least for porcine oocytes (61).
3. Electroporation system. The equipment needed for electroporation is an electropora-tion/electrofusion machine with the chamber. The requirements for such an apparatus are fairly simple, and a number of systems are available on the market that would be adequate for the purposes of oocyte activation. The machines most commonly used for such purposes are those manufactured by BTX (BTX has recently become part of Harvard Apparatus; Harvard Apparatus/BTX, Holliston, MA). The electropo-ration system should be able to deliver an adjustable ac current for dielectrophoretic aggregation of the cells. This is believed to be helpful during membrane fusion although in cases, when the AC current causes rotation of the reconstructed oocyte, manual alignment might be preferred. Alignment is normally followed by a variable DC square pulse to induce pore formation in the plasma membranes. The electrical output necessary depends primarily on the distance between the platinum or stainless steel electrodes of the electroporation chamber which most frequently is in the range of 0.5-1.0 mm. The duration and number of pulses should also be adjustable. After the DC current, AC reapplication may be helpful to keep cells together for more efficient fusion. An optional part of the BTX system is the "Optimizer," by which the user can verify whether the parameters delivered are actually those that were selected. In most situations, however, using the Optimizer is not essential. Recently, needle-type electrodes have also been developed (62). In this system the cell couplets are sandwiched between two wires attached to micromanipulators; the role of the manipulators is to make orientation easier and hence, fusion more effective.
4. The membrane destabilization evoked by the DC pulse also induces fusion of two cell membranes that are in direct contact. Because of this reason, electrofusion is frequently used to fuse the enucleated oocyte with the nuclear donor cell. In this case the reconstructed oocytes should be transferred one by one between the electrodes and aligned—either with an ac current or manually, using a fine capillary— so that the contact surface between the cytoplast and the donor cell is parallel to the electrodes. Then fusion and activation will be triggered with the same DC pulse.
5. There are contradictory reports about the necessity to prevent second polar body extrusion after activation. It generally is accepted that if NT takes place before DNA replication in the donor cell (G0-G: stage), extrusion of the second polar body after karyokinesis may result in the loss of chromosomes. Several possibilities exist for the prevention of the extrusion of the second polar body. Bovine oocytes incubated in the presence of cytochalasin B (the potent microtubule inhibitor) after a treatment with ionomycin completed the second meiotic division but did not extrude the second polar body resulting in the formation of multiple pronuclei. Alternatively, a 6-DMAP treatment after an induced calcium transient caused the second meiotic spindle to disintegrate and the oocytes progressed directly into interphase while forming only a single pronucleus (63). Finally phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C was also reported to inhibit second polar body extrusion. Incubation of electroporated porcine oocytes with PMA induced activation of protein kinase C that subsequently triggered inactivation of MAP kinase. MAP kinase inactivation ultimately caused the oocytes to go directly from metaphase into interphase without extruding the second polar body (64). However, inhibition of chromatin extrusion may not be beneficial if it occurs when the transferred nucleus is in G2. When large embryonic stem cells in the presumptive G2 stage were used to produce mice by means of NT, cytochalasin B was omitted from the culture medium following activation; it was present only when small cells (presumably prior to DNA replication) were used as nuclear donors (65). There also seems to exist an endogenous control mechanism in the oocyte: chromatin extrusion was seen in 81% of porcine oocytes receiving 4N somatic cell nuclei (those in G2 at the time of transfer) but in only 22% of oocytes receiving 2N nuclei (66). A treatment with cytochalasin B dissolved in DMSO is most commonly applied to prevent the extrusion of the second polar body. However, DMSO recently has been found to affect murine embryonic development after NT. Because of this reason, the use of ethanol as a solvent for cytochalasin B might be preferred for experiments whose purpose is to investigate nuclear-cytoplas-mic interactions (67). With the use of ethanol special care must be taken to avoid evaporation to maintain the desired concentration in the medium.
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