Zoltan Machaty

Somatic cell nuclear transfer in pigs: recent achievements and future possibilities

During the past 6 years, considerable advancement has been achieved in experimental embryology of pigs. This process was mainly generated by the rapidly increasing need for transgenic pigs for biomedical research purposes, both for future xenotransplantation to replace damaged human organs or tissues, and for creating authentic animal models for human diseases to study aetiology, pathogenesis and possible therapy. Theoretically, among various possibilities, an established somatic cell nuclear transfer system with genetically engineered donor cells seems to be an efficient and reliable approach to achieve this goal. However, as the result of unfortunate coincidence of known and unknown factors, porcine embryology had been a handicapped branch of reproductive research in domestic animals and a very intensive and focused research was required to eliminate or minimise this handicap. This review summarises recent achievements both in the background technologies (maturation, activation, embryo culture) and the actual performance of the nuclear replacement. Recent simplified methods for in vivo development after embryo transfer are also discussed. Finally, several fields of potential application for human medical purposes are discussed. The authors conclude that although in this early phase of research no direct evidence can be provided about the practical use of transgenic pigs produced by somatic cell nuclear transfer as organ donors or disease models, the future chances even in medium term are good, and at least proportional with the efforts and sums that are invested into this research area worldwide.

Capacitative Calcium Entry Mechanism in Porcine Oocytes

The presence of the capacitative Ca21 entry mechanism was investigated in porcine oocytes. In vitro-matured oocytes were treated with thapsigargin in Ca21-free medium for 3 h to deplete intracellular calcium stores. After restoring extracellular calcium, a large calcium influx was measured by using the calcium indicator dye fura-2, indicating capacitative Ca21 entry. A similar divalent cation influx could also be detected with the Mn21quench technique after inositol 1,4,5-triphosphate-induced Ca21 release. In both cases, lanthanum, the Ca21 permeable channel inhibitor, completely blocked the influx caused by store depletion. Heterologous expression of Drosophila trp in porcine oocytes enhanced the thapsigargin-induced Ca21 influx. Polymerase chain reaction cloning using primers that were designed based on mouse and human trp sequences revealed that porcine oocytes contain a trp homologue. As in other cell types, the capacitative Ca21 entry mechanism might help in refilling the intracellular stores after the release of Ca21 from the stores. Further investigation is needed to determine whether the trp channel serves as the capacitative Ca21 entry pathway in porcine oocytes or is simply activated by the endogenous capacitative Ca21 entry mechanism and thus contributes to Ca21 influx. calcium, embryo, fertilization, ovum

Time course of cortical and zona reactions of pig oocytes upon intracellular calcium increase induced by thimerosal.

Our previous study indicated that thimerosal is one of the most effective artificial activators to mimic sperm-induced increases in the intracellular free calcium concentration ([Ca2+]i) and other activation events in pig oocytes (Macháty et al., 1997). The present study was conducted to examine the temporal relationship between intracellular calcium transients, cortical granule (CG) exocytosis and the zona reaction induced by thimerosal. When pig oocytes matured in vitro were exposed to 200 microM thimerosal the first intracellular calcium transient, with a mean peak ratio of 4.97 +/- 1.14, was observed 509.64 +/- 122.03 s after addition of thimerosal. The density of CGs fell significantly from 63.3 +/- 11.7 CGs/100 micron 2 of cortex in control oocytes to 25.7 +/- 19.2 CGs/100 micron 2 of cortex (59.4% release) at 2 min after the first intracellular calcium transient. At 5 min after the calcium transient the residual CG density had been reduced to 10.7 +/- 10.4 CGs/100 micron 2 of cortex (83.1% release). This degree of CG exocytosis was the same as that in oocytes penetrated by sperm (9.5 +/- 5.1 CGs/100 micron 2 of cortex). No further decrease in residual CG density was observed at 10 min (10.3 +/- 14.8 CGs/100 micron 2 of cortex). Whereas 77.4% (120/155) of control oocytes were penetrated by spermatozoa only 1.4% (2/144) of thimerosal-treated oocytes were penetrated. Further experimental results obtained by in vitro fertilisation of oocytes with preincubated (capacitated) spermatozoa suggested that the zona block to sperm penetration in thimerosal-treated oocytes occurred within 35 min after CG exocytosis and 40 min after the first calcium transient. These results indicate that polyspermic penetration of pig oocytes inseminated in vitro is not due to delayed or incomplete CG exocytosis but more likely to a delayed zona reaction and/or simultaneous sperm penetration.

Orai1 mediates store-operated Ca2+ entry during fertilization in mammalian oocytes.

The presence of the store-operated Ca(2+) entry channel Orai1 and its function in signal transduction during fertilization have been investigated in mammalian oocytes using the pig as a model. RT-PCR cloning and sequence analysis revealed that Orai1 is expressed in the oocytes with a coding sequence of 921bp. After indirect immunocytochemistry or the overexpression of EGFP-tagged Orai1, the fluorescent signal was present primarily in the cell cortex consistent with plasma membrane localization of the protein. Western blot and real-time PCR results showed that Orai1 expression decreases during oocyte maturation; this is associated with the oocytes gaining the ability to generate a large Ca(2+) influx after store depletion. Downregulation of Orai1 expression by siRNA microinjection blocked Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the Ca(2+) oscillations induced by the fertilizing sperm were also inhibited in oocytes with downregulated Orai1 levels. At the same time, overexpression of Orai1 in the oocytes also modified store-operated Ca(2+) entry and had an inhibitory effect on the fertilization Ca(2+) signal. The abnormal Ca(2+) signaling due to Orai1 downregulation had a strong negative impact on subsequent embryo development. Co-overexpression of Orai1 and STIM1 on the other hand, led to a dramatic increase in Ca(2+) entry after store depletion. The findings indicate that Orai1 is a plasma membrane-resident Ca(2+) channel that is responsible for mediating Ca(2+) entry after the mobilization of intracellular Ca(2+) in oocytes. Orai1 and a functional store-operated Ca(2+) entry pathway are required to maintain the Ca(2+) oscillations at fertilization and to support proper embryo development.

STIM1 is required for Ca2+ signaling during mammalian fertilization.

During fertilization in mammals, a series of oscillations in the oocytes intracellular free Ca(2+) concentration is responsible for oocyte activation and stimulation of embryonic development. The oscillations are associated with influx of Ca(2+) across the plasma membrane that is probably triggered by the depletion of the intracellular stores, a mechanism known as store-operated Ca(2+) entry. Recently, STIM1 has been identified in oocytes as a key component of the machinery that generates the Ca(2+) influx after store depletion. In this study, the involvement of STIM1 in the sperm-induced Ca(2+) oscillations and its significance in supporting subsequent embryo development were investigated. Downregulation of STIM1 levels in pig oocytes by siRNA completely inhibited the repetitive Ca(2+) signal triggered by the fertilizing sperm. In addition, a significantly lower percentage of oocytes cleaved or formed blastocysts when STIM1 was downregulated prior to fertilization compared to the control groups. Restoring STIM1 levels after fertilization in such oocytes by means of mRNA injection could not rescue embryonic development that in most cases was arrested at the 2-cell stage. On the other hand, STIM1 overexpression prior to fertilization did not alter the pattern of sperm-induced Ca(2+) oscillations and development of these fertilized oocytes up to the blastocyst stage was also similar to that registered in the control group. Finally, downregulation of STIM1 had no effect on oocyte activation when activation was stimulated artificially by inducing a single large elevation in the oocytes intracellular free Ca(2+) concentration. These findings suggest that STIM1 is essential for normal fertilization as it is involved in the maintenance of the long-lasting repetitive Ca(2+) signal.

STIM1 regulates store-operated Ca2+ entry in oocytes.

The single transmembrane-spanning Ca(2+)-binding protein, STIM1, has been proposed to function as a Ca(2+) sensor that links the endoplasmic reticulum to the activation of store-operated Ca(2+) channels. In this study, the presence, subcellular localization and function of STIM1 in store-operated Ca(2+) entry in oocytes was investigated using the pig as a model. Cloning and sequence analysis revealed the presence of porcine STIM1 with a coding sequence of 2058 bp. In oocytes with full cytoplasmic Ca(2+) stores, STIM1 was localized predominantly in the inner cytoplasm as indicated by immunocytochemistry or overexpression of human STIM1 conjugated to the yellow fluorescent protein. Depletion of the Ca(2+) stores was associated with redistribution of STIM1 along the plasma membrane. Increasing STIM1 expression resulted in enhanced Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the influx was inhibited when the oocytes were pretreated with lanthanum, a specific inhibitor of store-operated Ca(2+) channels. When STIM1 expression was suppressed using siRNAs, there was no change in cytosolic free Ca(2+) levels in the store-depleted oocytes after Ca(2+) add-back. The findings suggest that in oocytes, STIM1 serves as a sensor of Ca(2+) store content that after store depletion moves to the plasma membrane to stimulate store-operated Ca(2+) entry.

Parthenogenetic Activation of Porcine Oocytes After Nuclear Transfer

Mature porcine oocytes are arrested at metaphase II of meiosis. At fertilization, like all mammalian oocytes they exhibit a low frequency Ca(2+) oscillation lasting several hours. This oscillation is thought to be the signal that triggers resumption of meiosis and activates the developmental program of the oocyte. The signal transduction mechanism of the sperm-induced Ca(2+) signal is not known in detail, and attempts to generate the oscillation artificially have met with little success. Nevertheless, artificial activation of the oocyte is a crucial step during nuclear transfer. Methods are available to induce a transient elevation in the intracellular free Ca(2+) concentration to surpass the meiotic arrest and induce development of the constructed embryo. Further studies concentrating on the mechanism of Ca(2+) signaling during fertilization will help to improve the efficiency of the procedures used for parthenogenetic activation of the oocyte.

Lipid characterization of individual porcine oocytes by dual mode DESI-MS and data fusion

The development of sensitive measurements to analyze individual cells is of relevance to elucidate specialized roles or metabolic functions of each cell under physiological and pathological conditions. Lipids play multiple and critical roles in cellular functions and the application of analytical methods in the lipidomics area is of increasing interest. In this work, in vitro maturation of porcine oocytes was studied. Two independent sources of chemical information (represented by mass spectra in the positive and negative ion modes) from single oocytes (immature oocytes, 24-h and 44-h in vitro matured oocytes) were acquired by using desorption electrospray ionization-mass spectrometry (DESI-MS). Low and mid-level data fusion strategies are presented with the aim of better exploring the large amount of chemical information contained in the two mass spectrometric lipid profiles. Data were explored by principal component analysis (PCA) within the two multi-block approaches to include information on free fatty acids, phospholipids, cholesterol-related molecules, di- and triacylglycerols. After data fusion, clearer differences among immature and in vitro matured porcine oocytes were observed, which provide novel information regarding lipid metabolism throughout oocyte maturation. In particular, changes in TAG composition, as well as increase in fatty acid metabolism and membrane complexity were evidenced during the in vitro maturation process. This information can assist the improvement of in vitro embryo production for porcine species.

Signal transduction in mammalian oocytes during fertilization

Mammalian embryo development begins when the fertilizing sperm triggers a series of elevations in the oocyte’s intracellular free Ca2+ concentration. The elevations are the result of repeated release and re-uptake of Ca2+ stored in the smooth endoplasmic reticulum. Ca2+ release is primarily mediated by the phosphoinositide signaling system of the oocyte. The system is stimulated when the sperm causes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG); IP3 then binds its receptor on the surface of the endoplasmic reticulum that induces Ca2+ release. The manner in which the sperm generates IP3, the Ca2+ mobilizing second messenger, has been the subject of extensive research for a long time. The sperm factor hypothesis has eventually gained general acceptance, according to which it is a molecule from the sperm that diffuses into the ooplasm and stimulates the phosphoinositide cascade. Much evidence now indicates that the sperm-derived factor is phospholipase C-zeta (PLCζ) that cleaves PIP2 and generates IP3, eventually leading to oocyte activation. A recent addition to the candidate sperm factor list is the post-acrosomal sheath WW domain-binding protein (PAWP), whose role at fertilization is currently under debate. Ca2+ influx across the plasma membrane is also important as, in the absence of extracellular Ca2+, the oscillations run down prematurely. In pig oocytes, the influx that sustains the oscillations seems to be regulated by the filling status of the stores, whereas in the mouse other mechanisms might be involved. This work summarizes the current understanding of Ca2+ signaling in mammalian oocytes.

Calcium influx in mammalian eggs

Calcium (Ca(2)(+)) signals are involved in the regulation of oocyte maturation and play a critical role during fertilization. In the egg, Ca(2)(+) is stored in the lumen of the endoplasmic reticulum and a signal is generated when the stored Ca(2)(+) is released through specialized channels in the membrane of the endoplasmic reticulum to elevate the free Ca(2)(+) concentration in the cytoplasm. Extracellular Ca(2)(+) is also important, indicated by the fact that the mobilization of luminal Ca(2)(+) is typically followed by Ca(2)(+) entry across the plasma membrane. The transmembrane Ca(2)(+) flux replenishes the endoplasmic reticulum, and thus, it is essential to sustain prolonged Ca(2)(+) signals. It also seems to be responsible for the stimulation of important signaling cascades required for complete egg activation. Characterization of the pathway that mediates Ca(2)(+) entry implies that its major components include STIM1, a protein that senses the filling status of the stores, and ORAI1, a channel protein located in the plasma membrane. Defining the mechanism and functions of Ca(2)(+) entry will not only lead to a better understanding of egg physiology but may also help improving the efficiency of a number of assisted reproductive technologies.