To examine the effects of somatic cell support on the cleavage and viability of fertilized sheep eggs, 434 pronucleate eggs were co-cultured for 3 or 6 days on oviduct cells or fibroblasts and 77 eggs were cultured in medium alone. During the first 3 days in culture 95% of the single-celled eggs cleaved regularly to non-compacted morulae on either of the feeder-layers but only 13% underwent similar regular cleavage in medium alone. Despite the identical cleavage rates in the co-culture groups, only 33% of embryos grown on fibroblasts as compared with 80% of embryos grown on oviduct cells were fully viable as judged by their ability to develop normally after transfer to recipient animals. The viability of embryos in the oviduct group was equal to that obtained after the direct transfer of morulae from donor to recipient sheep.After 6 days in culture 42% of embryos co-cultured with oviduct cells developed into expanded blastocysts as compared with only 4\m=.\5%cultured on fibroblasts. In both co-culture groups virtually all the remaining embryos blocked during the 4th cleavage. When transferred, 30% of blastocysts grown from the pronucleate stage on oviduct cells were viable.We conclude that: (1) during the first 3 days after fertilization cleavage will progress at a normal rate on different feeder-layers but oviduct cells appear to be required for the acquisition of full embryonic viability; (2) in our system oviduct cells are able to support passage of embryos through the critical 4th cell cycle, whilst fibroblasts are almost entirely unable to support this critical phase; and (3) it is apparent that the factors necessary for the morphological formation of the blastocyst may be insufficient or different from those which endow it with subsequent developmental ability.

The purpose of this work was to determine the mechanisms regulating the acquisition of cytoplasmic maturation and embryonic developmental competence in pig oocytes. The presence or the absence of porcine follicular fluid (pff; 25% or 0%) in the maturation medium was used as a means to achieve complete nuclear maturation accompanied or not accompanied by cytoplasmic maturation. ATP content, active mitochondria relocation, and microtubule distribution were analyzed at different times during in vitro maturation (IVM). While nuclear maturation did not differ among the two groups, parthenogenetic embryonic development was significantly higher (41.5%) in the 25% pff group than in the 0% pff group (19.0%) with blastocysts that had a significantly higher number of blastomeres (76.1 +/- 6.3, and 47.2 +/- 6.5, respectively). Oocyte ATP content increased significantly during IVM, but at the end of maturation no significant differences were observed between high- and low-competence oocytes. An extensive relocation of mitochondria to the inner cytoplasm during IVM together with the formation of a well-developed mesh of cytoplasmic microtubules was observed only in the high-competence oocyte group. However, no differences in the formation of microtubules associated with the meiotic spindles were observed between high- and low-competence groups. We conclude that low developmental competence is associated with the lack of a microtubule cytoplasmic network, which prevents correct relocation of mitochondria and is likely to reflect a more generally altered compartmentalization of the ooplasm. This can be independent from the formation of the microtubule machinery required for the completion of chromosome disjunctions and does not affect the overall ATP content.

At present, oocyte selection is mainly based upon morphological criteria but it is generally acknowledged that its reliability requires further improvement. The aim of this study was to determine whether transcript levels in cumulus cells can provide a useful marker of oocyte developmental competence in vitro. A retrospective study was performed on cumulus cells isolated from 90 oocytes retrieved from 45 patients. Upon fertilization, 35 oocytes originated good-quality embryos and 36 developed into poor-quality embryos, whereas 19 failed to be fertilized. Semi-quantitative measurement of hyaluronic acid synthase 2 (HAS2), gremlin1 (GREM1), and pentraxin 3 (PTX3) mRNAs was performed and data for all genes were obtained from all the samples. Cumulus cells isolated from oocytes that originated high-quality embryos on day 3 of culture had HAS2 and GREM1 transcript levels higher than those detected in cells from oocytes that did not fertilize or developed into poor-quality embryos. No differences were observed in PTX3 levels. Results indicate that the measurement of HAS2 and GREM1 levels in cumulus cells would reliably complement the morphological evaluation providing a useful tool for selecting oocytes with greater chances to be fertilized and develop in vitro.

In mammals, the study of gene expression in the preimplantation embryo has been difficult because the standard procedures used to quantify mRNA generally require large amounts of starting material. The development of protocols using different quantitative strategies generally involving the polymerase chain reaction (PCR) has provided new tools for exploration of gene expression in preimplantation embryos. However, the use of an internal standard, often referred as a housekeeping gene, is essential to normalize the mRNA levels. RNA levels of eight housekeeping genes were quantified using real time PCR throughout the preimplantation period of the bovine embryo to find the most suitable gene to be used as standard. Histone H2a was the best internal standard because the transcript levels were constant across the preimplantation period. Linear amplification of antisense RNA using the T7 promotor for in vitro transcription of the entire RNA pool was evaluated as a suitable way to preamplify the starting material prior to quantification and was effective in providing accurate RNA abundance profiles throughout the preimplantation period. However, the amplification appears to be template dependent because the amplification factors were higher for some genes.

The differentiated state of mature cells of adult organisms is achieved and maintained through the epigenetic regulation of gene expression, which consists of several mechanisms including DNA methylation. The advent of induced pluripotent stem cell technology enabled the conversion of adult cells into any other cell type passing through a stable pluripotency state. However, indefinite pluripotency is unphysiological, inherently labile, and makes cells prone to culture-induced alterations. The direct conversion of one cell type to another without an intermediate pluripotent stage is also possible but, at present, requires the viral transfection of appropriate transcription factors, limiting its therapeutic potential. The aim of this study was to investigate whether it is possible to achieve the direct conversion of an adult cell by exposing it to a demethylating agent immediately followed by differentiating culture conditions. Adult human skin fibroblasts were exposed for 18 h to the DNA methyltransferase inhibitor 5-azacytidine, followed by a three-step protocol for the induction of endocrine pancreatic differentiation that lasted 36 d. At the end of this treatment, 35 ± 8.9% fibroblasts became pancreatic converted cells that acquired an epithelial morphology, produced insulin, and then released the hormone in response to a physiological glucose challenge in vitro. Furthermore, pancreatic converted cells were able to protect recipient mice against streptozotocin-induced diabetes, restoring a physiological response to glucose tolerance tests. This work shows that it is possible to convert adult fibroblasts into insulin-secreting cells, avoiding both a stable pluripotent stage and any transgenic modification.pancreatic beta cell | cell plasticity R egenerative medicine requires new cells that can be delivered to patients for repairing and renovating degenerated or damaged tissues (1). When such cells are not readily available, two main strategies have been developed to obtain them: directed differentiation, by which pluripotent cells, exposed to specific cell culture conditions designed to mimic natural events, assume a specific cell fate, and transdifferentiation, also referred to as reprogramming, which enables a fully differentiated cell type to be converted into another without going through an undifferentiated pluripotent stage (2).Induced pluripotent stem cell (iPSC) technology showed that the stability of a mature phenotype can be overcome when transforming a somatic cell of any patient in an unlimited source of autologous pluripotent cells. The elimination of the immune rejection risk provided by iPSCs immediately boosted the clinical potential of directed differentiation (3). However, the requirement of permanent integration of viral vectors into the host genome to generate iPSCs poses a severe limit to their current therapeutic use (1). This has stimulated the development of several protocols for a virus-free iPSC derivation, but at present, these approaches are generally more technically demanding and l...