The development of somatic nuclear transfer procedures and the factors that are likely to affect the success of nuclear transplantation were discussed in the first chapter. In chapter 2, the effects of cell cycle stage and age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages and the in vitro life span of these cell lines were analyzed. The cell cycle characteristics of both fetal and adult cells were analyzed as a population of cells in culture. To study the individual cells from a population, a “shake off” method was developed to isolate G1 cells and evaluate progression through cell cycle. Irrespective of the age of the cell donor, the mean cell cycle length in isolated cells was shorter than what was observed for cells cultured as a population. In vitro development was analyzed after fusing confluent and isolated G1 cells to enucleated metaphase II oocytes. These results indicate that there were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells and confluent cells. Because previous studies do not clearly demonstrate the cell cycle stage of the donor cell that resulted in development to term, Chapter 2 evaluates the methods of producing populations of G0 and G1 cells and the cell cycle characteristics of G0 and G1 cells. Furthermore, in vitro and in vivo development of embryos derived from high confluent, G0 cells and “shake off” G1 cells were evaluated. These results indicate that the isolated G1 cells supported development to term better than confluent cells. Finally, in Chapter 3, pregnancy establishment, pregnancy loss and health of newborn calves were evaluated for nuclear transfer embryos derived from cells of different genotypes from dairy and beef breeds. In addition, the effect of overnight shipping of cloned embryos and the synchrony between the age of the embryo and the estrus cycle of the recipient female on development were assessed. The results indicate that genotype has an effect on development of nuclear transfer embryos.

In Chapter 1 we characterized the frequency, amplitude and duration of (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises in fertilized rabbit eggs loaded with fura-2 dextran. Their amplitude decreased and duration increased as fertilization progressed. Injection of 1,4,5 inositol trisphosphate (InsP3; IICR) or addition of thimerosal elicited (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises which were blocked by heparin, an InsP3 receptor antagonist. Ryanodine did not evoke Ca$\sp{2+}$ release. These results indicate that IICR is likely stimulated during fertilization. Fertilization (Ca$\sp{2+}\rbrack\sb{\rm i}$ changes were examined in in vitro matured bovine eggs (Chapter 2). Fertilized eggs exhibited different intervals between (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises which ranged from 15 min to more than 60 min. Unfertilized eggs were responsive to InsP3 injection and thimerosal exposure, although the frequency of the (Ca$\sp{2+}\rbrack\sb{\rm i}$ responses was shorter than the periodicity observed during fertilization. The mechanisms that generate (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations were examined in fertilized rabbit eggs (Chapter 3). Heparin blocked or delayed (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations. Oscillating eggs exhibited Ca$\sp{2+}$ release in response to CaCl2 injection. In unfertilized eggs, injection of GTP (S) induced (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations and enhanced the response to CaCl2 injection. Injection of InsP3 or CaCl2 elicited full Ca$\sp{2+}$ responses that reset the periodicity of ensuing oscillations. Thus, IICR participates in the generation of (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations and the unloading of the stores does not explain the interval between (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises. The signaling pathways possibly stimulated by the sperm during fertilization were investigated in unfertilized bovine eggs (Chapter 4). Injection of GTP (S) or InsP3 evoked (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations. Preinjection of heparin blocked sperm-mediated egg activation. Thimerosal elicited (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations which were not inhibited by heparin or ryanodine. The data suggest that bovine eggs possess a GTP-linked phosphoinositide pathway which appears to be stimulated by the sperm during fertilization. In Chapter 5 the amplitude and duration of (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises elicited by electrical pulses of different strengths and duration administered in variable extracellular Ca$\sp{2+}$ concentrations was reported. As these parameters increased, so did the peak (Ca$\sp{2+}\rbrack\sb{\rm i}$ elicited by the pulse. Young and aged eggs exhibited similar (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises when stimulated with identical pulses. However, their activation rates were different. Thus, aged eggs are more sensitive to a given (Ca$\sp{2+}\rbrack\sb{\rm i}$ rise.

This study evaluated procedures to improve culture and cryopreservation of in vitro produced bovine embryos. Successful in vitro development of early embryos is crucial for areas of biotechnology such as transgenics and cloning. The limiting factor in culturing in vitro matured and fertilized (IVM-IVF) embryos is the media in which the embryos develop in vitro. An optimal culture system which would support development equivalent to that found in vivo has yet to be found. If such a system were developed, studies could be conducted to elucidate the factors responsible for normal embryonic development and then apply these factors toward the development of defined media. The mitogenic capabilities of bovine (BVH) and rabbit (RVH) vitreous humor were tested on the in vitro development of IVM-IVF bovine embryos. Embryos were cultured from day 2, 3, 4, 5 or 6 until day 8 in different concentrations of BVH/RVH. Bovine embryos are inconsistent in their ability to develop in these fluids until the late morulae or early blastocyst stage. The activity of vitreous humor is not species-specific as bovine embryos developed in RVH as well or better than BVH. Blastocyst development was not improved with culture in BVH or RVH, although there is evidence to suggest that RVH enhances mean cell number in developing embryos. Delaying culture in BVH or RVH did improve mean cell number and blastocyst development. A simple and reliable method was developed to cryopreserve in vitro produced bovine embryos. The developmental capability of IVM-IVF derived morulae/early blastocysts was evaluated following different methods of diluting cryoprotectants after vitrification. One-step 0.1M or 0.3M sucrose dilution was optimal for removing the high concentrations of intracellular cryoprotectants in the vitrification medium as post-treatment survival was no different than culture controls. Step-wise dilution/gradual rehydration of vitrified embryos does not improve development following vitrification. Freezing and vitrification are equivalent for in vitro development of IVM-IVF bovine embryos following cryopreservation, although blastocyst development is approximately 1/2 the rate of their culture controls. These data are the first to indicate that IVM-IVF generated bovine embryos can be cryopreserved by vitrification.

The objective of the first series of experiments was to assess biochemical changes during in vitro maturation of oocytes collected from ovaries of adult cattle and calves (<6 mo old). Activity and/or concentrations of maturation-promoting factor, mitogen-activated protein kinase, and inositol 1,4,5-trisphosphate receptor were determined and they were significantly lower in calf oocytes. Developmental competence of parthenogenic embryos was studied after reciprocal transfer of metaphase II chromosomes between cow and calf oocytes and transfer of cumulus cells into cow and calf ooplasts. Development was higher in embryos originating from adult ooplasts. Collectively, these results support the hypothesis that lack of developmental competence of calf oocytes is due to their failure or inability to complete ooplasmic maturation. We hypothesized that the germinal vesicle (GV) stage oocyte was capable of inducing a meiotic like division of the somatic cell nucleus. To test this hypothesis, cumulus cells in G0, G1 or metaphase (M) were transferred to GV stage bovine oocytes. Denuded GV oocytes were fused by an electrical pulse and matured in vitro. Chromosome segregation was assessed after staining with Hoechst 33342. All cell stages had similar fusion and survival rates and were capable of undergoing a meiotic-like division on subsequent maturation. Human and mouse fibroblasts and bovine cumulus cells in G1 were transferred to GV or prometaphase I (PM I) stage bovine oocytes. Human and mouse fibroblasts were also transferred to PM 1 mouse oocytes. Meiotic maturation was assed by staining DNA with Hoechst 333342 and chromosome segregation by FISH analysis for bovine chromosomes 6 and 13 and for human chromosomes X and 18. Fusion and survival rates were better when PM I oocytes were used as recipients. Somatic cells were capable of undergoing a meiotic-like division. FISH analysis for reconstructed bovine oocytes revealed just 1 chromosome 6 and 13 per nuclei in 3 of 6 oocytes. After inter-specific nuclear transfer, chromosomes never completed meiotic segregation. These results support the hypothesis that haploidization of somatic cells can be induced after homologous transfer of nuclei into immature oocytes and is influenced by stage of oocyte development.

The program of a differentiated somatic cell can be altered by nuclear transplantation, cell fusion or co-culture. This work demonstrates that somatic gene function can be manipulated in vitro. We developed two novel strategies by which reprogramming of gene expression in a differentiated somatic cell can take place. Our first approach relies on the establishment of connections through gap junctions between somatic cells and blastomeres. We show that mouse epithelial cells are capable of forming gap junctions with blastomeres following injection into cleavage stage mouse embryos. In contrast, fibroblasts adhere but do not form gap junctions with blastomeres. Adhesion of the somatic cells to blastomeres was assessed by electron microscopy and immunological procedures using cell adhesion markers. Transfer of a fluorescent soluble dye from somatic cells to the adherent blastomeres demonstrated formation of functional gap junctions. Establishment of connections between epithelial cells and blastomeres correlated with induction of expression of the embryonic and ES cell-specific transcription factor, Oct-4, in the epithelial cells. We argue that communication between epithelial cells and blastomeres results in changes in somatic cell gene expression. Our second approach relies on the production of cell extracts to which permeabilized fibroblasts are exposed. The resulting fibroblasts are reprogrammed and thus take on characteristics and functions typical of the cell type from which the extract was derived, including the induction of Oct-4. The development of these reprogramming systems can be used to define the factors necessary for reprogramming somatic cells. Ultimately, these strategies for altering genome function could be applied to develop cell therapeutics.

Cloning of animals by nuclear transplantation has demonstrated that reprogramming of nuclear function is possible. However, low pregnancy rates, elevated pregnancy losses and lethal abnormalities in most cloned animals born argue that somatic cell reprogramming by nuclear transplantation is not always complete. Here, we report the identification of four nuclear markers of incomplete reprogramming in nuclear transplant mouse embryos. Nuclear transplant embryos exhibit (i) pronucleur assembly of A-type lamins, (ii) increased NuMA content, (iii) stronger anchoring of AKAP95 and (iv) a greater proportion of heterochromatin, compared to fertilized embryos. We propose that deficiencies in reprogramming through nuclear transplantation result from failure to morphologically remodel the somatic donor nucleus into a normal, fully functional pronucleus.