Showing papers by "Fulvio Gandolfi published in 2005"

Role of Adenosine Triphosphate, Active Mitochondria, and Microtubules in the Acquisition of Developmental Competence of Parthenogenetically Activated Pig Oocytes

Abstract: 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.

Cellular and molecular mechanisms regulating oocyte quality and the relevance for farm animal reproductive efficiency

Abstract: Summary The efficiency of breeding schemes is dependant on the high fecundity of the selected individuals. Reproductive technologies are constantly pushing the physiological limits, but while the male reproductive potential is almost fully exploited, female reproductive physiology is the subject of constant research. Since the number of offspring that a female can bring to term each pregnancy cannot be changed, the ideal approach is to remove the potential offspring at the beginning of development and to transfer them to recipients of lesser genetic value. The earlier the collection takes place, the higher the number of descendants that a female can generate, so that now, the number of available oocytes becomes the limiting factor. This article will describe how detailed studies on oocyte physiology are beginning to unravel the complex sequence that transforms a small primordial follicle into a large ovulatory follicle containing a mature oocyte. Progressively, the limits to oocyte manipulation have been recognised and gradually overcome with adequate hormonal treatments in vivo and with specific media supplementation in vitro. This has lead to the development of highly efficient reproductive technologies and the promise of even greater advances in the future. Surprising new findings, such as ovarian stem cells that can replenish the follicle population or long term embryonic stem cell lines that can differentiate into oocytes, are rapidly changing our expectations.

Effects of endocrine disrupters on the oocytes and embryos of farm animals.

Abstract: Currently, approximately 60 chemicals have been identified as endocrine disruptors (EDs): exogenous agents that interfere with the synthesis, secretion, transport, metabolism, binding, action, or elimination of natural blood-borne hormones. Farm animals ingest these substances with food and drinking water. Their stability and lipid solubility has led to increased concern that these substances may compromise the reproductive health of both humans and animals. Oocytes are a permanent cell population established before birth which is exposed to environmental stimuli for a period that, in farm animals, can be as long as several years. Oocyte competence is acquired within the ovary during the developmental stages that precede ovulation and its role is critical during the interval between fertilization and the so-called maternal to embryonic transition, when the transcriptional activity of the embryonic genome becomes fully functional. Any perturbation of these delicate process is likely to reduce oocyte developmental competence and, therefore, to cause an arrest of embryonic development at any given stage. A critical analysis of the doses and time of exposure is presented together with a description of the effects of different EDs on farm animal oocytes and early embryonic development. Finally some of the mechanisms mediating EDs effects on the oocytes will be described. In particular the role of arylhydrocarbon receptor, maternal mRNA stability and cytoplasmic remodelling during oocyte maturation will be discussed in some details.

168 establishment and molecular characterization of pig parthenogenetic embryonic stem cells

Abstract: Parthenogenetic embryonic stem cells have been obtained in mouse and in primates. However, it would be desirable to have an alternative experimental model that could be used to investigate the therapeutic potential of these cells. For this purpose, we generated parthenogenetic pig blastocysts from in vitro-matured oocytes activated by sequential exposure to 10 μM ionomycin for 5 min and 2 mM 6-DMAP for 3 h. Inner cell masses were isolated by immunosurgery and plated on mitotically inactivated STO fibroblast feeder layers in 4-well dishes. Cells were incubated in 5% CO2 at 37°C in low glucose DMEM/F10 medium supplemented with 1000 IU/mL of mouse recombinant LIF, 10% Knockout serum replacer (Gibco, Italy), and 5% FBS. Within 3 days, circular colonies with distinct margins of small round cells were observed on both substrates. When a colony enlarged enough to cover half or more of the well surface, cells were trypsinized in clumps never reaching single-cell suspension and passaged to a newly prepared well. The expression of a gene panel was examined by RT-PCR on a portion of the cells at each passage. Oct-4 and nanog were used as markers of pluripotency. Interferon-τ, α-Amilase, Bone Morphogenetic Protein-4, and Neurofilament were used as markers of trophectoderm, endoderm, mesoderm, and ectoderm differentiation respectively. After 4 passages, three colonies expressed Oct-4 and nanog and were negative for all four differentiation markers. Two colonies at the 5th and 7th passages maintained nanog but not Oct-4 expression, while remaining negative to all of the other genes. To induce the formation of embryoid bodies (EBs), cells were cultured in 50-μL droplets of medium without LIF. Initiation of differentiation of EBs was confirmed through both morphological examination and molecular analysis; mesodermal, ectodermal, and endodermal markers were all expressed by Day 9 of culture and Oct-4 and nanog expression was completely down-regulated. Interestingly, when EBs were returned to adherent culture conditions patches of differentiated cells tended to form, spontaneously differentiating into mesodermal, endodermal, or neuroectodermal cell monolayers. The present data suggest that it is possible to establish putative embryonic stem cells from pig parthenotes. Further studies are in progress to determine their ability to stably maintain the undifferentiated state. This work was supported by MIUR COFIN 20022074357 and Fondazione CARIPLO.