In vitro development and cytological quality of inter-species (porcine→bovine) cloned embryos are affected by trichostatin A-dependent epigenomic modulation of adult mesenchymal stem cells.

Artificial epigenomic modulation of in vitro cultured mesenchymal stem cells (MSCs) by applying a non-selective HDAC inhibitor, termed TSA, can facilitate more epigenetic reprogramming of transcriptional activity of the somatic cell-descended nuclear genome in NT pig embryos. The results of the present investigation showed that TSA-dependent epigenomic modulation of nuclear donor MSCs highly affects both the in vitro developmental capability and the cytological quality of inter-species (porcine→bovine) cloned embryos. The developmental competences to reach the blastocyst stage among hybrid (porcine→bovine) nuclear-transferred embryos that had been reconstructed with bovine ooplasts and epigenetically modulated porcine MSCs were maintained at a relatively high level. These competences were higher than those noted in studies by other authors, but they were still decreased compared to those of intra-species (porcine) cloned embryos that had been reconstituted with porcine ooplasts and either the cell nuclei of epigenetically transformed MSCs or the cell nuclei of epigenetically non-transformed MSCs. In conclusion, MSCs undergoing TSA-dependent epigenetic transformation were used for the first time as a source of nuclear donor cells not only for inter-species somatic cell cloning in pigs but also for inter-species somatic cell cloning in other livestock species. Moreover, as a result of the current research, efficient sequential physicochemical activation of inter-species nuclear-transferred clonal cybrids derived from bovine ooplasm and porcine MSC nuclei was developed.

Characterization of mesenchymal stem cells and their application in experimental embryology.

The efficiency of somatic cell cloning (somatic cell nuclear transfer; SCNT) as well as in vitro fertilization/in vitro embryo production (IVF/IVP) in mammals stay at relatively same level for over a decade. Despite plenty of different approaches none satisfactory break-through took place. In this article, we briefly summarize the implementation of mesenchymal stem cells (MSCs) for experimental embryology. The advantages of using MSCs as nuclear donors in somatic cell cloning and in vitro embryo culture are described. The description of results obtained with these cells in mammalian embryo genomic engineering is presented.

Interactions among activity of glucose-6-phosphate dehydrogenase in immature oocytes, expression of apoptosis-related genes Bcl-2 and Bax, and developmental competence following IVP in cattle.

This study was conducted to understand whether the level of G6PDH activity assessed in immature bovine oocytes by means of BCB test was correlated with the level of expression of apoptosis-related genes such as Bcl-2 and Bax in immature and mature oocytes. This information should support previous findings suggesting that G6PDH activity is a useful marker for determining oocyte quality, thereby increasing the validity of BCB test in oocyte selection. Up to now, there are no data estimating the relation between G6PDH activity and the expression of apoptosis-related genes in oocytes. The expression of Bcl-2 and Bax genes was estimated on the mRNA and protein levels, respectively using real-time PCR and Western-blotting. To evaluate developmental competence of these oocytes, cumulus-oocyte complexes classified as BCB+ (low activity of G6PDH), BCB- (high activity of G6PDH) and Control were used for in vitro embryo production. In immature oocytes, the Bax transcript level in BCB- oocytes was significantly higher (P<0.001) in comparison to Control. In mature oocytes, the Bcl-2 transcript level was significantly lower in BCB+ oocytes (P<0.01) and in BCB- oocytes (P<0.05) in comparison to Control. However, no relation was found between the activity of G6PDH and the expression of the Bcl-2 or Bax proteins, both in immature and mature oocytes. Our results on the transcript level seem to indicate that oocytes subjected to BCB staining show tendency towards apoptosis. However, results obtained at the protein level did not confirm this conclusion. The usefulness of the BCB test as the indirect marker of apoptosis seems to be questionable. The lack of significant differences in the blastocyst rates developed from BCB+ and Control oocytes decreases the validity of BCB test in IVP technology.

Effects of oocyte quality, semen donor and embryo co-culture system on the efficiency of blastocyst production in goats.

The aim of the study was to determine whether the selection of immature oocytes by a combination of cumulus-oocyte-complexes (COCs) morphology and staining with brilliant cresyl blue (BCB) would be helpful in selecting developmentally competent oocytes, and thereby increase the efficiency of blastocyst production from ovarian oocytes of FSH-primed, adult goats. In a second experiment the interaction between oocyte quality and semen donor was assessed. In a third experiment the usefulness of Vero cells for co-culture with goat embryos was investigated. In the pool of morphologically normal COCs recovered from ovaries following slicing (21.9+/-11.0), the mean rate of COCs classified as BCB+ was 85.6%, and the BCB- was approximately 11%. Oocytes classified as grade 1 and BCB+ exhibited the highest developmental competence (P<0.001) after in vitro maturation and fertilization compared with oocytes of grade 1 BCB- and grade 2 BCB+ or BCB-. There were no significant differences in developmental competence in grade 2 oocytes, regardless of BCB coloration. No significant differences in embryo cleavage and blastocyst formation rates among three bucks were observed when morphologically normal, BCB+ oocytes were used. For all tested bucks, differences in embryo production efficiency were related only to the oocyte quality. Similar blastocyst rates were developed from embryos co-cultured with goat oviduct epithelial cells (34.3%) and with Vero cells (33.3%). These results show that the most important criterion for selection of COCs before maturation is the visual assessment of morphological features. Staining with BCB of COCs recovered from adult goats does not enhance efficiency of selection of developmentally competent oocytes for IVF.