Following sperm-oocyte fusion, the Ca2+ signal triggered by the fertilizing sperm is responsible for stimulating meiotic resumption and eventually, embryonic development. In most cases, parthenogenetic activation methods also induce an elevation in the [Ca2+]i to activate the oocyte's developmental program . In many species numerous comparative studies have been carried out using different methods of artificial activation, and the most effective ones have been selected to activate reconstructed oocytes after nuclear transfer. In felids no such studies have been performed and the number of methods available for parthenogenetic activation of cat oocytes is rather limited. In the present study we characterized responses of cat oocytes to several artificial stimuli in order to find ways to trigger [Ca2+]i changes similar to those detected during mammalian fertilization. All the methods tested could elicit a rise in the [Ca2+]i of cat oocytes. Electroporation is commonly used to induce a transient elevation in the [Ca2+]i of oocytes of various species; it has also been successfully used to stimulate oocyte activation during cat nuclear transfer . The short, high voltage DC pulses are known to induce a significant transmembrane Ca2+ influx by causing a destabilization of the phospholipid bilayer. The influx is influenced by the voltage and duration of the electrical pulse as well as the ionic strength of the electroporation medium . We evaluated the effect of DC pulses with various duration and magnetic field characteristics; all pulses triggered changes in the [Ca2+]i. To stimulate embryonic development we have selected a set of parameters that induced a large [Ca2+]i elevation without causing irreversible damage to the oocytes.
Ethanol was reported to activate oocytes of various species. In Xenopus it was demonstrated to stimulate the production of inositol 1,4,5-trisphosphate (IP3) which induces the release of Ca2+ from the oocyte's intracellular stores . Its effect on cytoplasmic Ca2+ levels of cat oocytes has never been characterized before. We found that 7% ethanol triggered a long-lasting Ca2+ signal in cat oocytes as well. However, the effects of ethanol on subsequent embryonic development have not been tested in the present study because recently it was shown by others that ethanol could activate ~50% of cat oocytes . The Ca2+ ionophore ionomycin has also been shown to induce [Ca2+]i elevations in occytes of many species. According to the literature, the parameters of ionomycin treatment used to activate oocytes of various species vary significantly: concentrations ranging from 5 μM to 5 mM  and treatment duration of 1 min to 40 min have also been reported . The influence of ionomycin on cat oocytes has never been described; therefore optimizing the concentration and incubation time is critical for successful oocyte activation. As in other species, cat oocytes showed a larger elevation in the intracellular free Ca2+ levels after being treated with higher concentrations of ionomycin. Surprisingly, all the ionomycin concentrations that we tested for the ability to induce [Ca2+]i changes caused irreversible damages and were lethal to the oocytes.
Thimerosal is known to oxidize sulfhydryl groups on intracellular Ca2+ release proteins [35, 36], thus causing the release of stored Ca2+ and oscillation in the [Ca2+]i levels in oocytes of a number of species [24, 26, 37]. Similar to those findings, thimerosal in cat oocytes was able to induce repetitive [Ca2+]i changes. During incubation, the baseline [Ca2+]i became increasingly elevated between transients that was probably attributable to the inhibition of Ca2+-ATPases since it was reported that thimerosal not only induced Ca2+ release from the stores but also inhibited Ca2+-ATPases and prevented the removal of excess Ca2+ from the cytosol .
Incubation in the presence of Sr2+ induces activation in mouse oocytes  but the mechanism of its action has not been completely elucidated. Because in certain cell types Sr2+ is handled similar to Ca2+ it was proposed that Sr2+ activates oocytes by displacing bound Ca2+ . Recent studies showed that by using a low molecular weight heparin to antagonize the function of IP3 receptors or treating oocytes with the phospholipase C inhibitor U73122, the Sr2+-induced [Ca2+]i increases in mouse oocytes were blocked and this inhibitory effect could be rescued by microinjection of IP3. These results indicated that Sr2+ triggered the release of stored Ca2+ through the IP3 receptors . Although in mice Sr2+ treatment leads to an oscillation in the oocyte [Ca2+]i, we found that in cat oocytes Sr2+ triggered a single, long-lasting Ca2+ elevation.
The effects of Na+-free medium have been described in the pig where it was reported to induce oscillatory changes in the [Ca2+]i; . In approximately 50% of the cases, Na+-free medium also stimulated a [Ca2+]i elevation in cat oocytes with a high amplitude and long duration. The effect of the Na+-free medium is probably mediated by the Na+/Ca2+ exchanger expressed in the plasma membrane of cat oocytes. The Na+/Ca2+ exchanger uses the energy of a Na+ gradient to move Ca2+ . However, in case of low extracellular Na+ levels the exchanger can operate in a reverse mode, pumping Ca2+ into the cell. This feature of the exchanger makes Na+-free medium a potential candidate for stimulating oocyte activation. The exchanger was found to be present in hamster , mouse [44, 45] and porcine  oocytes. Because Na+-free medium triggered an elevation in the [Ca2+]i we decided to investigate its ability to stimulate parthenogenetic development in cat oocytes.
Based on the results of the fluorescence measurements, four different methods have been selected to activate cat oocytes. As a result of the treatments, pronuclear formation in the oocytes ranged between 50.9% and 67.7%. The combined thimerosal/DTT treatment followed by cycloheximide and cytochalasin B incubation led to blastocyst formation in 21.1% of the cases. This frequency is similar to that received after electroporation, the method most widely used for oocyte activation. Although thimerosal is able to induce Ca2+ oscillation in oocytes, it also oxidizes sulfhydryl groups on tubulin that prevents further development . In order to eliminate its negative effects, the thimerosal treatment must be followed by an incubation in the presence of the sulfhydryl-reducing compound DTT . The high percentage of blastocyst formation achieved in the present study indicates for the first time that the thimerosal/DTT activation is an effective way to stimulate parthenogenetic embryo development in cats.
Even though blastocyst formations induced by SrCl2 or Na+-free medium were somewhat low, this is the first report to demonstrate that these stimuli can trigger cat oocyte activation and subsequent blastocyst development. Although Na+-free medium has previously been reported to induce [Ca2+]i increases in porcine oocytes, no subsequent embryo development has been observed . Here we found that Na+-free medium could induce cleavage of cat oocytes with a frequency similar to that triggered by electroporation or the combined thimerosal/DTT treatment, although the subsequent blastocyst formation was lower compared to the other treatments. It is possible that the amplitude of the Ca2+ signal induced by the Na+-free medium is insufficient to induce complete egg activation. This is in accordance with the finding that in rabbit the amplitude of the Ca2+ transient did not appear to affect early cleavage but it influenced the developmental competence of the produced embryo . Another reason for the low blastocyst formation may be that sustained high levels of [Ca2+]i induced via the Na+/Ca2+ exchanger caused cellular damage that negatively influenced developmental competence. Although optimization of the Na+-free treatment was attempted in this study, the improvement in embryonic development was not significant. Additional studies focusing on the fine-tuning of this method may be helpful to develop a method that utilizes the Ca2+ signal generated via the Na+/Ca2+ exchanger for oocyte activation.
The results obtained also showed that incubation of cat oocytes in cycloheximide and cytochalasin B after an induced Ca2+ signal increased the frequency of pronuclear formation, cleavage and blastocyst formation. Mature mammalian oocytes arrested at the second metaphase stage synthesize cyclin B continuously in order to maintain activity of the M-phase Promoting Factor (MPF). During activation, MPF activity has to drop in order to allow release from the metaphase II arrest . Cycloheximide is a protein synthesis inhibitor that blocks the production of cyclin B and in turn decreases MPF activity. Cytochalasin B on the other hand is an inhibitor of actin polymerization frequently used to block the extrusion of second polar body. In the presence of cytochalasin B, segregation of the chromosomes occurs after parthenogenetic oocyte activation but cytokinesis does not take place which results in the formation of diploid zygotes with two pronuclei [22, 48]. In our experiments, incubation of cat oocytes with cycloheximide and cytochalasin B had the tendency to improve pronuclear formation, cleavage frequency and blastocyst development after every single Ca2+ elevation-inducing stimulus tested and the improvement was significant in several cases. Although the incubation did not increase the total cell number of the developing blastocysts, the results indicate that these inhibitors are beneficial to increase the efficiency of oocyte activation in the cat.
The objective of the oocyte activation experiments was to develop potential methods to be used during nuclear transfer for the generation of useful disease models. In the domestic cat, somatic cell nuclear transfer not only provides the opportunity to generate genetically identical animals for research purposes, it also has the potential to facilitate preservation of rare and valuable felid populations, including cat research models and possibly endangered felid species . In addition, domestic cats are useful research models for the study of more than 200 human hereditary diseases . Maintenance of cat disease models can be challenging however, because the disease state may interfere with normal breeding, gestation and/or parturition. The application of nuclear transfer technology offers an innovative approach. Cloning would eliminate the need to collect gametes from affected animals for in vitro fertilization, it would obviate the requirement to maintain populations of carrier (heterozygous) cats, and for cat models that do not survive to sexual maturity cloning would provide a direct method for their continued propagation. Based on the frequency of blastocyst formation they induced, two methods were selected to activate reconstructed cat oocytes after nuclear transfer. These methods included electroporation and the combined thimerosal/DTT treatment, each followed by incubation in the presence of cycloheximide and cytochalasin B.
For nuclear transfer, fibroblast cells from two animals affected by different metabolic defects were collected and used as nuclear donors. The defects included AMD and MPS I, two lysosomal storage diseases that have been extensively characterized in cats. They are homologous to human inborn errors, involve single gene mutations and inherited as autosomal recessive traits [49, 50]. These models have been instrumental in demonstrating the value of bone marrow transplantation in alleviating these diseases and, most recently, the potential of gene therapy using an adeno-associated viral vector for treating AMD-affected cats [14, 15]. Cats affected with MPS I are difficult to breed naturally due to musculoskeletal abnormalities whereas AMD-affected cats typically die before reaching puberty.
Transfer of cloned embryos in which development was stimulated with electroporation followed by cycloheximide and cytochalasin B incubation resulted in 5 females having distinct implantation sites and 4 females with established pregnancies (i.e., presence of at least one fetus). Despite a total of 8 implantation sites, 5 fetuses and detectable heartbeats in two fetuses, no term development occurred; all fetuses were eventually reabsorbed by day 49 of pregnancy. Our previous research has demonstrated the developmental competence to term of MPS-affected and MPS-carrier IVF embryos following transfer into our primary recipient type . Pregnancy loss in the current study may be attributable to deficient reprogramming of the donor nucleus in the enucleated oocyte or aberrant chromosome numbers as a result of incomplete enucleation. Furthermore, no pregnancies were detected following thimerosal/DTT-induced oocyte activation which was somewhat unexpected. Nevertheless, the thimerosal/DTT treatment stimulated cleavage divisions at a frequency comparable to that achieved by electroporation. This, in light of a recent report showing that the thimerosal/DTT activation supported term development of cloned transgenic piglets  implies that successful activation can be achieved by modification of sulfhydryl groups on key Ca2+ releasing proteins in cat oocytes.